1. Field of the Invention
The present invention relates to a method for producing L-cysteine or related substances. Specifically, the present invention relates to a bacterium suitable for the production of L-cysteine or related substances and a method for producing L-cysteine or related substances utilizing such a bacterium. L-cysteine and L-cysteine related substances are useful in the fields of drugs, cosmetics, and foods.
2. Brief Description of the Related Art
L-cysteine is conventionally obtained by extraction from keratin-containing substances such as hair, horns, and feathers, or by the conversion of DL-2-aminothiazoline-4-carboxylic acid using a microbial enzyme. L-cysteine has also been produced on a large scale by using an immobilized enzyme method and a novel enzyme. Furthermore, it has also been attempted to produce L-cysteine by fermentation utilizing a microorganism.
Microorganisms which are able to produce L-cysteine are known. For example, a coryneform bacterium with increased intracellular serine acetyltransferase activity produces cysteine (Japanese Patent Laid-open (Kokai) No. 2002-233384). The ability to produce L-cysteine can also be increased by incorporating serine acetyltransferase which has been mutated to attenuate feedback inhibition by L-cysteine (Japanese Patent Laid-open No. 11-155571, U.S. Patent Published Application No. 20050112731, U.S. Pat. No. 6,218,168).
Furthermore, the ability to produce L-cysteine in a microorganism can be enhanced by suppressing the L-cysteine decomposition system. Examples of such microorganisms include coryneform bacteria or Escherichia bacteria in which the activity of cystathionine-β-lyase (Japanese Patent Laid-open No. 11-155571), tryptophanase (Japanese Patent Laid-open No. 2003-169668), or O-acetylserine sulfhydrylase B (Japanese Patent Laid-open No. 2005-245311) is attenuated or deleted.
Furthermore, it is known that the ydeD gene which encodes the YdeD protein participates in secretion of the metabolic products of the cysteine pathway (Dabler et al., Mol. Microbiol., 36, 1101-1112 (2000)). Furthermore, techniques for enhancing the ability to produce L-cysteine by increasing expression of the mar-locus, emr-locus, acr-locus, cmr-locus, mex-gene, bmr-gene or qacA-gene are also known. These loci and/or genes encode proteins which cause secretion of toxic substances from cells (U.S. Pat. No. 5,972,663). emrAB, emrKY, yojIH, acrEF, bcr, and cusA are further examples (Japanese Patent Laid-open No. 2005-287333).
An Escherichia coli has been reported which produces L-cysteine, and which has increased activity of the positive transcriptional control factor of the cysteine regulon encoded by the cysB gene (International Patent Publication WO01/27307).
It is known that dsbA (Shevchik V. E., et al., EMBO J., 1994 Apr. 15; 13(8):2007-12) participates in the formation of disulfide bonds (S—S) of proteins as described below, but the relation thereof with L-cysteine production is not known.
In general, many proteins with a disulfide bond (S—S) are among proteins that are secreted into cell membrane surface layers or out of cells. The interior of cells enclosed by the cell membranes is under relatively mild conditions both physically and chemically. However, since the exterior of cells is a more severe environment, the structures of proteins need to be more rigid. The presence of S—S bonds in proteins is one of the strategies.
Anfinsen et al. demonstrated that oxidative folding of proteins accompanied by the formation of S—S bonds spontaneously advanced under moderate oxidative conditions (Givol D. et al., J. Biol. Chem., 1964, Sep.; 239:PC3114-16). However, it has been elucidated that, in a cell, there is a delicate system for the formation of S—S bond, so that it can advance more quickly and correctly as compared with the aforementioned formation of S—S bonds (Bardwell J. C., et al., Cell, 1991 Nov. 1; 67(3):581-9.). For example, in the periplasm of Escherichia coli, a series of proteins called Dsb factors (disulfide bond, DsbA, DsbB, DsbC (Shevchik V. E., et al., EMBO J., 1994 Apr. 15; 13(8):2007-12), DsbD (Missiakas D., et al., EMBO J., 1995 Jul. 17; 14(14):3415-24), and DsbG (Andersen C. L., et al., Mol. Microbiol., 1997, Oct.; 26(1):121-32)) form disulfide bonds and introduce them into many proteins. In the periplasm, DsbA (disulfide oxidoreductase) oxidizes a substrate protein (AmpC, DsbB, LivK, OstA, RcsF (Kadokura H., et al., Science, 2004, Jan. 23; 303(5657):534-7)), and the cysteine pair of DsbA converted into the reduced type is re-oxidized with the intracellular membrane protein DsbB. It has been elucidated that electrons received by DsbB are transferred to ubiquinone (UQ), which is a respiratory chain component, and oxygen eventually serves as an electron acceptor with the aid of cytochrome oxidase (Inaba K., et al., J. Biol. Chem., 279, 6761-6768 (2004)).
Although DsbA has superior characteristics as an S—S bond-introducing enzyme, the ability thereof to select a cysteine pair into which the bond is introduced is not sufficient. It has also been found that the enzyme DsbC (disulfide isomerase) which recombines incorrect S—S bonds is present in the periplasm of Escherichia coli. In order for DsbC to function as an isomerase, the active site thereof must be maintained as a reduced type, contrary to DsbA, which is an oxidase. It is considered that the source of the reducing power originates in the thioredoxin system existing in the cytoplasm, and the reducing power from thioredoxin is transferred to DsbC via an intracellular membrane protein DsbD (Rietsch A., et al., J. Bacteriol., 1997 November; 179(21):6602-8). Moreover, it is also considered that a protein called DsbG which shows narrower substrate specificity is also present in Escherichia coli, and it has activities of both disulfide oxidoreductase and disulfide isomerase (Andersen C. L., et al., Mol. Microbiol., 1997, Oct.; 26(1):121-32).
As for the use of the aforementioned Dsb family, there are such findings as mentioned below, with expecting the effect of correctly forming S—S bonds of proteins. Many proteins derived from eukaryotic organisms have a disulfide bond, and they cannot be expected to have their original structures when they are expressed in the cytoplasm of Escherichia coli, which is under severe reducing conditions. Therefore, it is considered that, for production of such proteins derived from eukaryotic organisms, expression in the form of secretion into the periplasm is effective, which is under an oxidative environment preferred for formation of disulfide bond. However, although many attempts of expressing heterogenous proteins by secretion into Escherichia coli periplasm have been reported, any heterogenous protein cannot necessarily be expressed as active proteins. This poses a problem especially for proteins having many disulfide bonds. Meanwhile, in Escherichia coli, the roles of DsbA, DsbB, DsbC, and DsbD, which are the Dsb family proteins involved in the formation of disulfide bond, were deduced on the basis of biochemical tests and complementation tests using strains deficient in each of the genes (Bardwell, J. C., Mol. Microbiol., 14, 199-205 (1994), Sone, M., et al., J. Biol. Chem., 272, 10349-10352 (1997), Rietsch, A., et al., Proc. Natl. Acad. Sci. USA, 93, 13048-13053 (1996)). Therefore, there have been made several attempts of overexpressing DsbA or DsbC together with an objective protein with the goal of improving the secretion expression of the objective protein into the periplasm (Japanese Patent Laid-open No. 2000-83670). Furthermore, it is also already known that DsbA works as a re-activating agent for proteins that do not have physiological activity (Akiyama Y., et al., J. Biol. Chem., 1992 Nov. 5; 267(31):22440-5, Japanese Patent Laid-open No. 5-336986). However, only the oxidation of proteins has been described in the reports to date, and there is no example of reporting involvement of the Dsb factors in oxidation of low molecules such as amino acids. Therefore, it has been completely unknown whether the Dsb factors oxidize low molecular weight molecules such as cysteine to form cystine.